Neuronal hyperexcitability and excitotoxicity are thought to underlie a number of clinical disorders. Unfortunately, very little is known about the mechanisms that lead to excessive neuronal activity. Strong indirect evidence indicates that neuronal excitability is influenced by small changes in [K+]o and that astrocytes are important in the regulation of [K+]o. Astrocytes are thought to maintain [K+]o within the narrow limits required for normal neuronal activity be removing K+ from areas of neuronal activity (a process referred to as spatial buffering). Spatial buffering is thought to be accomplished by the uptake of K+ through an inwardly rectifying K+ channel (Kir) and the dissipation of K+ into a syncytium of astrocytes connected by gap junctions. We propose to test the hypothesis that spatial buffering is dependent on the expression of astrocytic KirS and gap junctions and that elimination of these in astrocytes leads to increases in [K+]o, neuronal excitability and seizure activity. Cre/loxP technology and an inducible, astrocyte-specific gene expression system will be used to carry out conditional knockouts (cKO) of connexin43 (cn43) and Kir4.1; these proteins are necessary for gap junction communication (gjc) between and K+ uptake into astrocytes, respectively. The effects of eliminating astrocytic cn43 and KirS will be examined in situ using hippocampal brain slices and in vivo using models of hyperexcitability. The following questions will be addressed in these studies. First, in vivo, doe a cKO of cn43 or Kir4.1 lead to spontaneous seizures or an increased sensitivity to seizure- promoting stimuli? Second, does a cKO of cn43 or Kir4.1 lead to abnormal increases in [K+]o during neuronal activity in situ? Third, does a cKO of cn43 or Kir4.1 lead to spontaneous seizure- like activity or an increased sensitivity to seizure-promoting stimuli in hippocampal slices? And fourth, does a cKO of cn43 or Kir4.1 affect the efficacy or plasticity of synaptic transmission at the Schaffer collateral-CA1 pyramidal neuron synapse? It is our premise that while it is clear that astrocytes exhibit properties that would enable them to modulate neuronal activity in vivo, it is essential to develop model systems whereby the role of astrocytes in brain function and dysfunction can be examined. The goal of this proposal is to develop such a model system and to test the hypothesis that astrocytes regulate neuronal activity through their ability to modulate [K+]o.